Nutrient import across Gram-negative bacteria's outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB-ExbD-TonB. Having purified the ExbB4-ExbD2 complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB4-ExbD2 complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB4-ExbD2 was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB4-ExbD2 complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB6-ExbD4, and propose a structural arrangement of its transmembrane α-helical domains.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/s00232-014-9678-4 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Membrane Protein Complex ExbB4-ExbD1-TonB1 from Escherichia coli Demonstrates Conformational Plasticity.
J Bacteriol
June 2015
Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada Microbiome and Disease Tolerance Centre, McGill University, Montreal, QC, Canada
Unlabelled: Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted.
View Article and Find Full Text PDFAmphipol-trapped ExbB-ExbD membrane protein complex from Escherichia coli: a biochemical and structural case study.
J Membr Biol
October 2014
Department of Microbiology and Immunology, McGill University, Montreal, H3A 2B4, QC, Canada.
Nutrient import across Gram-negative bacteria's outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB-ExbD-TonB. Having purified the ExbB4-ExbD2 complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB4-ExbD2 complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy.
View Article and Find Full Text PDFCoordinated rearrangements between cytoplasmic and periplasmic domains of the membrane protein complex ExbB-ExbD of Escherichia coli.
Structure
May 2014
Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada; Microbiome and Disease Tolerance Centre, McGill University, Montreal, QC H3A 2B4, Canada. Electronic address:
Gram-negative bacteria rely on the ExbB-ExbD-TonB system for the import of essential nutrients. Despite decades of research, the stoichiometry, subunit organization, and mechanism of action of the membrane proteins of the Ton system remain unclear. We copurified ExbB with ExbD as an ∼240 kDa protein-detergent complex, measured by light scattering and by native gels.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!